Otitis media is the most common illness of early childhood with approximately 70% of all children suffering at least one bout of otitis media before the age of seven. Chronic otitis media can lead to hearing, speech and cognitive impairment in children. It is caused by bacterial infection with Streptococcus pneumoniae (approximately 50%), non-typable Haemophilus influenzae (approximately 30%) and Moraxella (Branhamella) catarrhalis (approximately 20%). In the United States alone, treatment of otitis media costs between one and two billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. Because otitis media occurs at a time in life when language skills are developing at a rapid pace, developmental disabilities specifically related to learning and auditory perception have been documented in youngsters with frequent otitis media.
B. catarrhalis mainly colonizes the respiratory tract and is predominantly a mucosal pathogen. Studies using cultures of middle ear fluid obtained by tympanocentesis have shown that B. catarrhalis causes approximately 20% of cases of otitis media. (ref. 1-Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure.)
The incidence of otitis media caused by B. catarrhalis is increasing. As ways of preventing otitis media caused by pneumococcus and nontypeable H. influenzae are developed, the relative importance of B. catarrhalis as a cause of otitis media can be expected to further increase.
B. catarrhalis is also an important cause of lower respiratory tract infections in adults, particularly in the setting of chronic bronchitis and emphysema (refs. 2, 3, 4, 5, 6, 7, and 8). B. catarrhalis also causes sinusitis in children and adults (refs. 9, 10. 11, 12, and 13) and occasionally causes invasive disease (refs. 14, 15, 16, 17, 18, and 19).
Like other Gram-negative bacteria, the outer membrane of B. catarrhalis consists of phospholipids, lipopolysaccharide (LPS), and outer membrane proteins (OMPs). Eight of the B. catarrhalis OMPs have been identified as major components. These are designated by letters A through H, beginning with OMP A which has a molecular mass of 98 kDa to OMP H which has a molecular mass of 21 kDa (ref. 20).
Of the major OMPs identified in B. catarrhalis an apparent doublet, named CD, is a heat modifiable protein. It has a molecular mass of 55 kDa at room temperature and a mass of 60 kDa when heated under reducing conditions. This protein is surface exposed and conserved among a variety of B. catarrhalis strains, as demonstrated by a study using CD-specific monoclonal antibodies (ref. 21). The gene encoding CD was recently cloned and expressed in E. coli by Murphy et al (ref. 22). Restriction mapping of 30 isolates of B. catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays, indicating the sequence of the CD gene to be highly conserved.
Thus, the heat-modifiable protein CD of B. catarrhalis is a surface exposed, conserved protein that contains at least two epitopes that are present in all studied strains of B. catarrhalis (ref. 21). Properties of CD protein indicate that the protein has utility in diagnosis of and vaccination against disease caused by B. catarrhalis or other bacterial pathogens that produce CD protein or produce a protein capable of raising antibodies specifically reactive with CD protein.
It would be advantageous to provide purified CD protein (and methods of purification thereof) for use as antigens, immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents.